Alkylating agents are an important class of anticancer drugs, which express their cytotoxic and antitumour effects by forming adducts with cellular DNA.
Bifunctional nitrogen mustard alkylating agents such as chlorantbucil, melphalan, and cyclophosphamide are a major subset of this class of drugs. Their mechanism of antitumour action has been shown to be via interstrand cross-linking of cellular DNA, primarily at guanine N7 sites in runs of guanines in the major groove, which are both the most accessible and the most nucleophilic DNA sites. Because the two alkylating functions on the nitrogen mustard are in such close proximity, cross-linking is limited to DNA sequences which contain two reactive nucleophilic centers within reach of the mustard, with most of the interstrand cross-links being between proximate guanines. Due partly to these spatial restraints, a large proportion of the molecules alkylate DNA only once, forming monoadducts which are primarily genotoxic rather than cytotoxic. A major mechanism of cellular resistance to nitrogen mustards is increased DNA repair of the crosslinks which are formed.
There has been less work on compounds designed to alkylate in the minor groove of DNA, where the most susceptible sites are the N3 of adenine and the exocyclic amino group of guanine, which are the sites targeted respectively by the two best known minor groove alkylating agents CC-1065 (Hurley et al., 1988) and anthrarnycin (Hurley & Needham-VanDevanter, 1986). These compounds are extraordinarily-potent cytotoxins, in spite of forming only monoadducts, possibly because they do not readily induce DNA repair enzymes (Tang et al., 1988). There is also recent evidence, using transcription termination assays with linearized plasmid DNA containing the 420 base pair Pst 1 fragment of exon 2 of the human c-myc oncogene, that two nitrogen mustards (chlorambucil and melphalan) cause termination of transcription preferentially at adenines (at every adenine pair in the melphalan-treated template, and at selected A-G and GA pairs in the chlorambucil-treated template), in spite of the fact that most of the alkylation by these compounds occurs at guanines (Pieper et al., 1989).
For these reasons we have been interested in the development of minor groove-targeted bifunctional alkylating agents as potential antitumour drugs. We report here the design and synthesis of a family of spatially-separated bis-mustards, and studies on interaction with DNA and antitumour properties of a representative compound.
While there have been recent reports on the preparation of bifunctional aniline mustard analogues of the minor groove binding polypyrrole antibiotic distamycin A, we are not aware of any examples of spatially-separated minor groove-targeted mustards. However, a recent report on compounds containing two CC-1065 alkylating units showed that these compounds cross-linked DNA and displayed extraordinary cytotoxic potency (Mitchell et al, 1989).
It was decided to employ aniline mustards as the alkylating moleties in the design of compounds of this invention because of their well-understood alkylation chemistry and because their reactivity can be modulated over a wide range by suitable choice of substituents in the aromatic ring (Palmer et al., 1990). Although there has been little evidence to date of the ability of aniline mustards to alkylate DNA in the minor groove, they have not been designed to target this site. This is despite the fact that the aniline ring of such compounds can itself form part of the minor groove-targeting ligand. The design of was based on the polybenzamide bisquaternary ammonium heterocycles (Denny et al., 1979; Braithwaite and Baguley, 1980), with the dimethylaminomethyl group replacing the terminal quaternary ammonium rings.